IE74901B1 - Glycopeptide antibiotics derived from balhimycin - Google Patents

Abstract

Demethylbalhimycin, a compound of the formula C65H71Cl2N9O24, demethylleucylbalhimycin, a compound of the formula C59H60Cl2N8O23, deglucobalhimycin, a compound of the formula C60H63Cl2N9O19, ureidobalhimycin, a compound of the formula C67H74Cl2N10O25, demethyldeglucobalhimycin, a compound of the formula C59H61Cl2N9O19, and balhimycin V, a compound of the formula C73H84Cl2N10O26, methylbalhimycin, a compound of the formula C67H75Cl2N9O24, balhimycin R, a compound of the formula C72H83Cl2N9O28, have an antibiotic action.

Description

The present invention relates to novel glycopeptides, a process for their preparation and their use.

A large number of glycopeptide antibiotics have already been described. However, many of these antibiotics are more weakly active than the original type of glycopeptides and commercial product vancomycin and are inferior to this, in particular also in vivo (cf. R. Nagarajan Antimicrobial Agents and Chemotherapy, April 1991, pages 605-609).

Vancomycin may indeed be employed in infectious diseases which are caused by gram-positive pathogens, but a number of severe side effects, such as, for example, the socalled red man syndrome, sphacelation and others greatly restrict the applicability. Another very active glycopeptide antibiotic is balhimycin (cf. EP 0 468 504, to which reference is expressly made at this position).

It has now surprisingly been found that strongly active antibiotic substances can be made available with the compounds related to balhimycin, the side effects known from vancomycin not occurring or occurring in diminished form.

The invention accordingly relates to desmethylbalhimycin, a compound of the formula I, HO OH OH desmethyl-desglucobalhimycin, a compound of the formula V, methylbalhimycin, a compound of the formula Ce7H75Cl2N9O24, 5 balhimycin R, a compound of the formula C72H83C12N9O28 and balhimycin V, a compound of the formula C73H84Cl2N10O26 and their hydrates and physiologically tolerable salts.

The hydrates of the compounds mentioned are formed by addition of water, such as shown below by the example of desmethylbalhimycin.

HO OH OH C0NH2 CH (CH3) g Physiologically tolerable salts of the compounds mentioned are, for example, the acetates, hydrochlorides, phosphates, sulfates, etc., which can be obtained in a generally known manner.

The present invention furthermore includes the process for the preparation of the compounds mentioned. A process for the preparation of the compounds mentioned is characterized in that the microorganism Actinomyces species Y-86,21022 (DSM 5908) is cultured in an aqueous nutrient medium and the target compounds are then isolated and purified. The microorganism mentioned has been deposited on April 6, 1990 under the conditions of the Budapest Convention.

The microorganism mentioned is cultured as described in the abovementioned EP in an aqueous nutrient medium containing carbon sources, nitrogen sources and mineral salts. Preferred culturing conditions are described in the abovementioned EP; further preferred conditions are mentioned in the example below.

During culturing of the microorganism mentioned, balhimycin and, in only small amounts, the abovementioned compounds are mainly formed. By variation of the nutrient base composition, in particular with respect to the nitrogen source, the formation of distinctly larger amounts of the compounds according to the invention can be achieved. Thus, it has surprisingly been found that the addition of millimolar concentrations of methionine, serine and pyruvate suppresses the formation of balhimycin. If methionine antagonists, such as, for example, ImM α-methylmethionine, are employed, a distinct increase in the yields takes place with greater emphasis on the desmethyl component of balhimycin. Allosteric inhibitors of aspartate metabolism, such as L-lysine or L-threonine and leucine antagonists, likewise have an effect on the product spectrum.

Moreover, the product spectrum of the strain Actinomyces species Y-86,21022 can be affected by genetic measures. Mutations with mutagens of physical or chemical type known per se in combination with suitable selection methods, for example antimetabolite resistance, lead to mutants which produce the desired secondary components in greatly increased amounts or exclusively.

The separation of the glycopeptides mentioned is preferably carried out by means of cation exchangers in buffer systems having a high content of organic solvents. Suitable solvents are, for example, water-miscible organic solvents such as lower alcohols, acetone, acetonitrile, glycol, dioxane, dimethyl sulfoxide, formamide and the like, but also aqueous urea solutions. Preferred solvents are methanol, ethanol, isopropanol· and acetone. Particularly suitable solvent contents are 5-95% of organic solvents in the aqueous buffer solutions, particularly preferred contents are between 25 and 85%. Since the separation effect somewhat improves with increasing solvents content, the separation is expediently carried out in practice with a content of organic solvent of not below 35%.

Another possibility for separation on a scale which can be carried out industrially consists in using reverse phases and improving the sharpness of separation by suitable measures. Such measures are the use of additives such as salts, for example phosphate buffer and others or of chaotropic substances such as urea, KC1O4 or other agents such as complexing agents and ion-pairing agents, inter alia, in the eluents.

An alternative step for the isolation of the compounds according to the invention is crystallization. In this case, the tendency of the compounds according to the invention to crystallize in the vicinity of the isoelectric points, and their dependance on solvent admixtures in the mother liquor and on the type of counter ions is utilized. For example, compounds according to the invention in aqueous solution can be brought to crystallization by addition of water-soluble organic solvents such as, for example, ethanol or isopropanol.

Alternatively, the compounds present in aqueous acidic solution are brought to crystallization by increasing the pH, for example by means of addition of NH3. The crystalline compounds obtained, such as, for example, ureidobalhimycin, which crystallizes in the non-centrosymmetrical space group Pl with 2 molecules in the elemental cell and the cell constants a - 17.909 A, b = 18.466 A, c = 18.873 A, a = 96.65, β = 114.15, γ = 114.78°, also belong to the present invention.

Another process for the preparation of desmethylleucylbalhimycin consists in carrying out an Edman degradation with balhimycin or with desmethylbalhimycin (cf. Practical Protein Chemistry, A Handbook A. Darbre, page 345 ff., John Wiley & Sons, 1987).

An additional process for the preparation of desglucobalhimycin comprises carrying out a hydrolytic cleavage with balhimycin. A particularly preferred hydrolysis agent is 4N or more highly concentrated trlfluoroacetic acid, in particular even at slightly elevated temperature.

An additional process for the preparation of desmethyldesglucobalhimycin comprises carrying out a hydrolytic cleavage with desmethylbalhimycin. A particularly preferred hydrolysis agent is 4'N or more highly concentrated trifluoroacetic acid, in particular at room temperature or slightly elevated temperature.

An additional process for the preparation of ureidobalhimycin comprises reacting balhimycin with isocyanates such as, for example, potassium isocyanate or with urea.

This reaction can be carried out, for example, in aqueous solution within a wide pH range, preferably in the range between pH 4 and 8.

The novel compounds according to the invention are closely related to the glycopeptide antibiotic balhimycin and are structurally derived from this. They can be characterized in detail as follows: a) Desmethylbalhimycin is formed by the strain Y-86,21022 (DSM 5908) and has the following properties: Empirical formula: ^-65^71^2^9024 determined by FAB mass spectrometry: M + H+ = 14 32.4 for the isotope: 12Ci51H7135Cl214N916O24 Chemical molecular weight: 1433.25 Da Amino acid analysis (after hydrolysis in 5 M hydrochloric acid at 100°C, 20 hours): Aspartic acid, leucine, besides other unusual ninhydrin-positive substances UV maxima: 281 nm (log E 3.8) Desmethylbalhimycin thus differs from balhimycin in that it contains leucine instead of N-methylleucine. b) Desmethylleucylbalhimycin is produced by means of the strain Y-86,21022 (DSM 5908) and has the following properties: Empirical formula: C59H60Cl2N8O determined by FAB mass spectrometry: M + H+ = 1319.3 for the isotope: 12C591H6035Cl214N816O24 Chemical molecular weight: 1320.08 Da Amino acid analysis (after hydrolysis in 5 M hydrochloric acid at 100°C, 20 hours): Aspartic acid, in addition to unusual ninhydrin-positive substances.

Absent: leucine and N-methylleucine UV maxima: 281 nm (log E 3.8) Desmethylleucylbalhimycin differs from balhimycin by Nmethylleucine being absent. c) Desglucobalhimycin is formed by the Actinomycetes strain Y-86,21022 (DSM 5908), and has the following properties: Empirical formula: Ci0H63Cl2N9O19 determined by FAB mass spectrometry: M + H+ = 1284.4 for the isotope: 12C601H6335C1214Nb16O19 Chemical molecular weight: 1285.12 Da Amino acid analysis (after hydrolysis in 5 M hydrochloric acid at 100°C, 20 hours): Aspartic acid, N-methylleucine in addition to unusual ninhydrin-positive substances .

UV maxima: 279 nm (log e 3.8) Desglucobalhimycin differs from balhimycin by the absence of a glucose residue. d) Ureidobalhimycin is formed from the Actinomycetes strain Y-86,21022 (DSM 5908) and has the following properties: Empirical formula: C67H-74Cl2N10O determined by FAB mass spectrometry: M + H+ = 1489.4 for the isotope: 12C671H7435C214N1016O25 Chemical molecular weight: 1490.29 Da UV maxima 280 nm (log e:3.8) Ureidobalhimycin is the cyclic ureide of the antibiotic balhimycin on carbon atoms 3 and 4 of dehydrovancosamine. e) Methylbalhimycin is formed from Actinomyces strain Y-86,21022 (DSM 5908) and has the following properties: Empirical formula: ^-67H75Cl2N9O24 determined by FAB mass spectrometry: 5 M + H+ = 1460.45 for the isotope: 12C671H7535C1214N916O24 f) Balhimycin R is formed from Actinomyces strain Y-86,21022 (DSM 5908) and has the following properties: Empirical formula: C72H83Cl2N9O2e determined by FAB mass spectrometry: M + H+ = 1592.48 for the isotope: 12C721H8335C1214N916O24 Chemical molecular weight: 1593.41 Da.

UV maxima 280 nm (log e = 3.8) Balhimycin R differs from balhimycin by an additional rhamnosyl radical. g) Desmethyl-desglucobalhimycin is formed from Actinomyces strain Y-86, 21022 (DSM 5908) and has the following properties: Empirical formula: C5SH61C12N9O19, determined by FAB mass spectrometry: M + H+ = 1270.35 for the isotope: 12C591H6135C1214N916O19.

Chemical molecular weight: 1271.09 UV maxima 280 nm (log e = 3.8). h) Balhimycin V is formed from the Actinomyces strain Y-86, 21022 (DSM 5908) and has the following properties: Empirical formula: ^73®84^12Ν10Ο determined by FAB mass spectrometry: M + H+ = 1587.50 for the isotope: 12C731H8435Cl2N10O26 - 12 Chemical molecular weight: 1588.44 Da.

UV maxima 280 nm (log e = 3.8).

Balhimycin V differs from balhimycin by an additional 4-dehydrovancosaminyl radical.

The compounds according to the invention are colorless substances soluble in water or in aqueous solutions, which are comparatively surprisingly stable in the form of a solid or in solution.

The following Table 1 shows some biological data: Minimum bacteriostatic inhibitory concentrations in micrograms per milliliter determined by means of the Agar dilution method: Table 1 Desmethyl- balhimycin Desmethyl- leucyl- Desgluco- balhimycin 15 Staph, aureus SG 511 0.1 balhimycin 3 0.2 Staph, aureus 285 0.1 6 0.2 Staph, aureus 503 0.05 6 0.1 Strept. pyogenes 308 A 0.05 12.5 0.1 20 Strept. pyogenes 77 A 0.05 3 0.1 Strept. faecium D 0.2 6 0.2 Escherichia coli DC 2 10 >100 >100 Bact. fragilis 312 100 50 25 Bact. fragilis 960 25 50 25 25 Bact. fragilis 1313 50 100 25 Bact. vulgatus 1446 50 100 >100 Peptostrept.anaeroh (.932 25 50 0.4 Propioni acnes 6916 0.8 6 0.2 Propioni acnes 6922 0.4 6 0.2 30 Clostridium tetani ATCC 1940650 Clostridium perfringens 194 50 0.2 50 6 0.4 0.1 Ureido- Methyl- Balhimycin Balhimycin balhimycin R Staph, aureus SG 511 0.8 0.4 0.2 Staph, aureus 285 1.5 0.4 0.8 5 Staph, aureus 503 1.5 0.4 0.2 Strept. pyogenes 308A 0.8 0.4 0.2 Strept. pyogenes 77 A 0.8 0.4 0.2 Strept. faecium D 1.5 0.8 0.4 Escherichia coli DC 2 >100 25 50 10 Bact. fragilis 312 25 100 >100 Bact. fragilis 960 25 100 >100 Bact. fragilis 1313 25 50 >100 Bact. vulgatus 1446 50 >100 >100 Peptostrept. anaerob.932 6.2 0.8 100 15 Propioni acnes 6916 0.8 0.4 6.2 Propioni acnes 6922 1.5 0.4 3.1 Clostridium tetani ATCC 1940650 Clostridium 194 12.5 0.4 3.1 1.5 100 20 perfringens Desmethyl- desgluco- balhimycin Balhimycin V Staph, aureus SG 511 0.2 0.4 25 Staph, aureus 285 0.2 0.4 Staph, aureus 503 0.1 0.1 Strept. pyogenes 308A 0.1 0.05 Strept. pyogenes 77 A 0.1 0.05 Strept. faecium D 0.2 0.2 30 Escherichia coli DC 2 > 100 >100 Bact. fragilis 312 50 n. t. Bact. fragilis 960 25 n. t. Bact. fragilis 1313 50 n. t. Bact. vulgatus 1446 > 100 n. t. 35 Peptostrept. anaerob.932 0.2 n. t. Propioni acnes 6916 0.2 n. t. Propioni acnes 6922 0.1 n. t.

Clostridium tetani ATCC 19406 Clostridium perfringens 194 n. t 0.1 n. t. n. t As can be seen from Table 1, the compounds according to the invention in particular have an outstanding action against gram-positive bacteria including the so-called methicillin-resistant Staphylococcus aureus strain (MRSA). They are therefore in particular suitable for the treatment of infectious diseases which have been caused by such microorganisms. The subject of the invention accordingly also includes pharmaceuticals containing an effective amount of a compound according to the invention and the use of the compounds for the preparation of pharmaceuticals, in particular of pharmaceuticals having antibiotic action; said preparation is carried out in a conventional, generally known manner.

The compounds according to the invention are furthermore also suitable for use as growth promoters in agriculture. The present invention will be illustrated in greater detail by means of the examples below and the contents of the patent claims.

Example 1: Fermentation of the balhimycin components The nutrient solution (NL 5276) is used as the main culture for the fermentation. It is composed as follows.

NL 5276: Glycerol, 99% g/1 of distilled water Soya peptone HySoy T Glucose CaCO3 Yeast extract, Oxoide pH before sterilization g/1 5 g/1 3 g/1 3 g/1 7.0 The substrates, apart from glucose, are added to 10 ml of water with stirring and made up to a volume of 18 1. The pH before sterilization is adjusted to 7.0 using dilute -30% strength NaOH. The amount of glucose in the mixture specified above is dissolved separately in 1 1 of water and the solution is sterilized for 20 minutes at 120°C in an autoclave and added to the sterilized mixture after cooling. Sterilization is carried out for 45 minutes at 120°C and 1.2-1.4 bar. After cooling to operating temperature and adding the glucose solution, the fermentation volume is about 20 1 with a pH of about 7.0. The C fermenter is inoculated with 500-1000 ml of preculture, which is prepared as in EP 0 468 504. Examples 2 and 3. > Fermentation conditions: Fermentation temperature 28°C Aeration 20 1/minute = 1 wm Pressure 0.5 bar Speed 250 rpm As an antifoaming agent, if corresponding to 0.025% relative volume, of Leverkusen) mixture. necessary, 5 ml, to the fermentation RDesmophen 3600 (polyols, Bayer AG, are added as a sterile water-Desmophen The fermentation time is 96-120 hours. The pH is not corrected during the fermentation but the culture is examined for sterility, nitrogen consumption and the formation of products by means of HPLC. It is then harvested and the cell material is removed by centrifugation.

Example 2: Isolation of the balhimycin complex 10 1 of filtrate from the cultures obtained as in Example 1 are added to a previously prepared column containing 1 1 of RDiaion HP-20 (Mitsubishi Chem. Ind.). The loaded support is then washed with demineralized water. The balhimycin components are then eluted with a gradient containing 0-50% of isopropanol and the outflow from the column is collected in fractions. The fractions are examined for antibiotic activity and the component composition is determined by means of HPLC. First, desmethylbalhimycin-containing fractions (I), then balhimycin-richer (II) and finally desmethylleucylbalhimycin-richer fractions (III) are obtained. They are collected separately and after concentration and freezedrying give 650 mg of I, 1.1 g of II and 380 mg of III.

Example 3: Isolation of desmethylbalhimycin by ion chromatography A 100 ml chromatography column is packed with RFractogel EMD-SO3 cation exchanger and adjusted to pH 4.8 (buffer A) with 25 mM sodium acetate buffer in 66% methanol. 650 mg of desmethylbalhimycin-containing antibiotic, obtained, for example, in accordance with Example 2 are then dissolved in approximately 100 ml of buffer A and applied to the column, and the latter is washed with 100 ml of buffer A. The antibiotic desdehydrovancosamine derivative known from the literature is found in the runnings and in the washing water.

Elution system: Detection: Retention time: A 0-200 mM sodium chloride gradient in buffer A, pH 5.0 is then applied. The balhimycin is eluted from the ion exchanger using 130-150 mM NaCl, and the desmethylbalhimycin using 160-175 mM NaCl solution. The corresponding fractions are each dialyzed against 1/100 M acetic acid and freeze-dried. Crystallization from aqueous solution with the addition of ethanol leads to 210 mg of balhimycin acetate in 98% purity and 160 mg of desmethylbalhimycin acetate in 97% purity.

High pressure liquid chromatography (HPLC) data: Support: RLichrospher RP18, 5 μιη, 250 x 4 mm2 14% acetonitrile in 0.1% strength aqueous trifluoroacetic acid UV absorption at 210 nm 8.3 minutes, comparison balhimycin: 10.0 minutes [«l"1 -77 ± 2° Example 4: Preparation of pure desmethylleucylbalhimycin 300 mg of the desmethylleucylbalhimycin-containing product III obtained as in Example 2 are additionally purified again on 100 ml of MCI gel CHP20P (Mitsubishi Chem. Ind.) in accordance with Example 2, but 10 mM K2HPO4 buffer, pH 7.6, is used as buffer A and 10 mM K2HPO4, pH 7.6 in 40% strength methanol is used as buffer B. The elution carried out in the gradient process gives fractions which are analyzed by means of HPLC. The desmethylleucylbalhimycin-containing fractions, having a purity of over 90%, are combined, concentrated in vacuo and desalted on reverse phase RP18, in a 0.05% trifluoroacetic acid/acetonitrile system. Freeze-drying of the main fractions gives 120 mg of desmethylleucylbalhimycin trifluoroacetate in 98% purity.

High pressure liguid chromatography (HPLC) data: Support: Eluent: Detection: Retention time: [«]d .

RLichrospher RP18, 5 pm, 250 x 4 mm2 14% acetonitrile in 0.1% strength aqueous trifluoroacetic acid UV absorption at 210 nm 16.0 minutes, comparison balhimycin: 10.0 minutes +27 + 2° Example 5: Obtaining desglucobalhimycin and desmethyldesglucobalhimycin 300 mg of the product III obtained as in Example 2 are dissolved in water and applied to a preparative 500 ml capacity HPLC column (250-2), which is packed with the support RNucleosil 1015 C18 P (Macherey-Nagel, Duren). The latter is then eluted in the gradient process with 0-20% acetonitrile in 0.1% trifluoroacetic acid. While the antibiotics balhimycin and desmethylleucylbalhimycin are first dissolved from the support using an 8-10% solvent content, desmethyl-desglucobalhimycin and desglucobalhimycin are obtained using a 14-15% acetonitrile content. Freeze-drying of the desmethyl-desglucobalhimycin- or desglucobalhimycin-containing fractions and their rechromatography in the same system give 1.3 mg of desmethyl-desglucobalhimycin trifluoroacetate salt or mg of desglucobalhimycin trifluoroacetate salt respectively.

Example 6: Hydrolytic degradation of balhimycin to desglucobalhimycin g of balhimycin, obtained in accordance with the application EP 0 468 504, Example 4, are dissolved in 120 ml of 4 molar trifluoroacetic acid and allowed to react overnight at 45°C. After this period, the solvent is removed in vacuo and then by freeze-drying. The reaction mixture concentrated in this way is then dissolved in water and separated on 800 ml of MCI gel CHP20P (Mitsubishi Chem. Ind.) using the gradient system 0.1% acetic acid/0.1% acetic acid in 50% strength isopropanol. Balhimycin and desamidobalhimycin are first eluted from the column, then desglucobalhimycin and finally desamidodesglucobalhimycin. The desired fractions having a degree of purity of over 90% are collected, rechromatographed and freeze-dried. They give 1.3 g of desglucobalhimycinacetate in a purity of 98.5%.

High pressure liquid chromatography (HPLC) data Support: RLichrospher RP18, 5 gm, 250 x 4 mm2 Eluent: 19% acetonitrile in 0.1% strength aqueous trifluoroacetic acid Detection: UV absorption at 210 nm Retention time: 10.0 minutes [a] , -70.5 + 2° Example 7: Obtaining ureidobalhimycin g of crude balhimycin (II), obtained in accordance with Example 2, are dissolved in 25 ml of water, the pH is adjusted to 3.5 with acetic acid and the solution is applied to a previously prepared column containing 150 ml Fractogel EMD-SO3 cation exchanger equilibrated at pH 3.5.

After application, the column is first washed with 200 ml of pure water, then with 200 ml of 25 mM sodium acetate buffer pH 4.0 (buffer A).

The column outflow of this buffer solution contains the ureidobalhimycin, called eluate WP. The column is then eluted by applying a 0.1 M NaCl gradient in 25 mM sodium acetate, pH 4.0. Balhimycin R (eluate R) is obtained using 20-30 mM NaCl, mainly balhimycin (eluate B) using 30-70 mM NaCl and desmethyl- and methylbalhimycin (eluate M) using 80-100 mM NaCl.

For the preparation of pure ureidobalhimycin, the eluate WP is added to a nucleosil 10-C18 AB reverse phase column (20 mm ID x 250 mm length) and separated in the gradient process using the 0.1% trifluoroacetic acid/acetonitrile system - as outlined in Example 5. Freeze-drying of the ureidobalhimycin-containing fraction gives 20 mg of this antibiotic as the trifluoroacetate salt.

High pressure liquid chromatography (HPLC) data: Support: Eluent: Detection: Retention time: E«]£4= "Lichrospher RP18, 5 μπι, 250 x 4 mm2 14% acetonitrile in 0.1% strength aqueous trifluoroacetic acid UV absorption at 210 nm 13.5 min, comparison balhimycin 10.0 min -26° (c = 1% in water) Example 8: Obtaining methylbalhimycin The eluate M obtained as in Example 7 is purified on the 20 mm x 250 mm (ID χ H) "Nucleosil 10-C18 AB column with the aid of the gradient system 10 mM K2HPO4, pH 7.5/45% methanol in 10 mM K2HPO4, pH 7.5.

The column outflow is controlled by the analytical HPLC system - as described below - and the methylbalhimycincontaining fractions are combined, concentrated in vacuo and desalted by adsorption on "MCI gel CHP20P as in Example 6. Freeze-drying of the phosphate-free pure antibiotic solution gives 11 mg of methylbalhimycin9 acetate in 98% purity.

High pressure liquid chromatography (HPLC) data: Support: RLichrospher RP18, 5 μπι, 250 x 4 mm2 Eluent: Detection: Retention time: 14% acetonitrile in 0.1% strength aqueous trifluoroacetic acid UV absorption at 210 nm .8 min, in comparison to balhimycin: .0 min -59° (c = 1% in water) Example 9: Hydrolytic degradation of desmethylbalhimycin to desmethyl-desglucobalhimycin 100 mg of desmethylbalhimycin, obtained according to Example 3, are dissolved in 2 ml of 90% strength trifluoroacetic acid and allowed to react at room temperature for 70 hours. The mixture is then worked up according to Example 6. 72 mg of desmethyl-desglucobalhimycin acetate are obtained in 98% purity.

High pressure liquid chromatography (HPLC) data: Support: RLichrospher RP 18, 5μ, 250 x 4 mm2 Eluent: 19% acetonitrile in 0.1% strength Detection: Retention time: aqueous trifluoroacetic acid, UV absorption at 210 nm 9.1 min Example 10: Obtaining balhimycin R 150 mg of the desalted and freeze-dried eluate R obtained as in Example 7 are rechromatographed on the same Fractogel column as described in Example 7. The balhimycin R now obtained in 79% purity is purified and desalted further on reverse phase "Nucleosil 10 RPieAB as Example 7 - in the 0.1% trifluoroacetic acid system. The freeze-dried antibiotic (52 mg) is dissolved in 3 ml of water, the pH is slowly adjusted to 6 and a further 0.6 ml of ethanol is added to the solution after crystallization commences. After crystallization is complete, the mixture is centrifuged and the crystallizate is washed with ethanol and dried in vacuo. 22 mg of balhimycin R in 99% purity result.

High pressure liquid chromatography (HPLC) data: Support: Eluent: Detection: Retention time: RLichrospher RP18, 5 μπι, 250 x 4 mm2 14% acetonitrile in 0.1% strength aqueous trifluoroacetic acid UV absorption at 210 nm 7.9 min, comparison balhimycin: 10.0 min Example 11: Obtaining ureidobalhimycin from balhimycin 1500 mg of balhimycin obtained in accordance with EP 0 468 504 are dissolved in 60 ml of water, 162 mg of potassium cyanate are added, the pH is adjusted to 6 and the solution is allowed to stand for 2 hours. After this time, it is separated by preparative HPLC in the 0.1% trifluoroacetic acid system of a 500 ml capacity RNucleosil 1015 C18 P, 250-2 column. The ureidobalhimycin-containing fractions are collected separately from the balhimycin, freeze-dried and crystallized in water/ethanol at pH 5. Centrifugation and drying give 1.3 g of ureidobalhimycin in over 98% purity.

Example 12: Obtaining balhimycin V A 100 ml chromatography column is packed with RFractogel EMD-SO3 cation exchanger and equilibrated with 25mM ammonium formate buffer, pH 4.2 (buffer A). 1 g of crude balhimycin (II), obtained according to Example 2, is dissolved in 100 ml of water, the solution is adjusted to pH 4 and applied to the column, and the latter is washed with 200 ml of buffer A.

A 0.5 M sodium chloride gradient in buffer A, pH 4 is subsequently applied. The less basic antibiotics of the balhimycin series are eluted from the column first with a 0.34 - 0.36 M NaCl solution of balhimycin V. The corresponding balhimycin V-containing fractions are collected and desalted as described in Example 6 on 100 ml of RMC1 gel CHP 20 P and freeze-dried. They yield 80 mg of balhimycin V acetate.

Higher pressure liquid chromatography (HPLC) data: Support: Eluent: Detection: Retention time: RLichrospher RP, 18.5 μπι, 250 x 4 mm2 14% acetonitrile in 0.1% strength aqueous trifluoroacetic acid UV absorption at 210 nm .3 to 10.4 min, as the wider peak, comparison with balhimycin: 10 min Retention time of the reaction product of balhimycin V with potassium cyanate according to Example 10: 12.4 min Comparison with balhimycin: 10 min.

FAB mass spectrum: Mass calculated from all molecular ions 1588 Da.

ESI mass spectrum: Mass is calculated from all molecular ions.

MW 1588 Da (diketone form), MW 1606 Da (monohydrate), MW 1624 Da (dihydrate)

Claims (5)

CLAIMS:

1. Desmethylbalhimycin, a compound of the formula I, desmethylleucylbalhimycin, a compound of the formula 5 II, desglucobalhimycin, a compound of the formula III, ureidobalhimycin, a compound of the formula IV, desmethyldesglucobalhimycin, a compound of the formula V, V H <>< ^OH HO □H

2. . 2 .

3. . 3 .

4. . 5 . 20 6. 7. methylbalhimycin, a compound of the formula C S7 H 75 C ^2 N 9°24 Z balhimycin R, a compound of the formula C72H83Cl2NgO28 and balhimycin V, a compound of the formula C 73Ba4Cl 2 N 10 O 26 , and their hydrates and physiologically tolerable salts. A compound as claimed in claim 1 in crystalline form. A compound as claimed in claim 1, which can be prepared by fermentation of Actinomyces species Y-86,21022 (DSM 590 8) in an agueous nutrient medium and subsequent isolation. A process for the preparation of compounds as claimed in claims 1 to 3, which comprises culturing the microorganism Actinomyces species Y-86,21022 (DSM 5908) in an agueous nutrient medium and then isolating and purifying the target compounds. A process for the preparation of desmethylleucylbalhimycin by Edman degradation of balhimycin or of desmethylbalhimyc in. A process for the preparation of desglucobalhimycin by hydrolytic cleavage of balhimycin. A process for the preparation of ureidobalhimycin which comprises reacting balhimycin with an isocyanate or with urea. 8. A process for the preparation of desmethyldesglucobalhimycin by hydrolytic cleavage of desmethylbalhimycin . 9. A pharmaceutical containing an effective amount of at least one compound as claimed in claim 1 or 2. 10. The use of a compound as claimed in claim 1 or 2 for the production of pharmaceuticals. 11. The use of a compound as claimed in claim 1 or 2 for the production of pharmaceuticals having antibiotic action. 12. A process for the production of pharmaceuticals which comprises bringing a compound as claimed in claim 1 or 2 into a suitable administration form, if appropriate using pharmacologically acceptable auxiliaries and/or excipients . 13. The use of a compound as claimed in claim 1 or 2 as a growth promoter in agriculture. 14. A compound as claimed in claim 1, substantially as hereinbefore described and exemplified. 15. A process for the preparation of a compound as claimed in claim 1, substantially as hereinbefore described and exemplified. 16. A compound as claimed in claim 1, whenever prepared by a process claimed in any one of claims 4 - 8 or 15. 17 A pharmaceutical according to claim 9, substantially as hereinbefore described. 18. Use according to claim 10, substantially as hereinbefore described. 19. a process for the preparation of a pharmaceutical according to claim 9, substantially as hereinbefore described. 4 . 5 . 20 6. 7.

5. 20. A pharmaceutical according to claim 9, whenever prepared by a process claimed in claim 12 or 19.